Changes in Expression of Manganese Superoxide Dismutase, Copper and Zinc Superoxide Dismutase and Catalase in calyciflorus during the Aging Process

Jianghua Yang, Siming Dong, Qichen Jiang, Tengjiao Kuang, Wenting Huang, Jiaxin Yang* Jiangsu Province Key Laboratory for Biodiversity & Biotechnology and Jiangsu Province Key Laboratory for Aquatic Live Food, School of Biological Sciences, Nanjing Normal University, Nanjing, Jiangsu, People’s Republic of China

Abstract are useful model organisms for aging research, owing to their small body size (0.1–1 mm), short lifespan (6–14 days) and the relative easy in which aging and senescence phenotypes can be measured. Recent studies have shown that antioxidants can extend the lifespan of rotifers. In this paper, we analyzed changes in the mRNA expression level of genes encoding the antioxidants manganese superoxide dismutase (MnSOD), copper and zinc SOD (CuZnSOD) and catalase (CAT) during aging to clarify the function of these enzymes in this process. We also investigated the effects of common life- prolonging methods [dietary restriction (DR) and resveratrol] on the mRNA expression level of these genes. The results showed that the mRNA expression level of MnSOD decreased with aging, whereas that of CuZnSOD increased. The mRNA expression of CAT did not change significantly. This suggests that the ability to eliminate reactive oxygen (ROS) in the mitochondria reduces with aging, thus aggravating the damaging effect of ROS on the mitochondria. DR significantly increased the mRNA expression level of MnSOD, CuZnSOD and CAT, which might explain why DR is able to extend rotifer lifespan. Although resveratrol also increased the mRNA expression level of MnSOD, it had significant inhibitory effects on the mRNA expression of CuZnSOD and CAT. In short, mRNA expression levels of CAT, MnSOD and CuZnSOD are likely to reflect the ability of mitochondria to eliminate ROS and delay the aging process.

Citation: Yang J, Dong S, Jiang Q, Kuang T, Huang W, et al. (2013) Changes in Expression of Manganese Superoxide Dismutase, Copper and Zinc Superoxide Dismutase and Catalase in Brachionus calyciflorus during the Aging Process. PLoS ONE 8(2): e57186. doi:10.1371/journal.pone.0057186 Editor: Gabriele Sorci, CNRS, Universite´ de Bourgogne, France Received November 5, 2012; Accepted January 18, 2013; Published February 22, 2013 Copyright: ß 2013 Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Natural Science Foundation of China (No.: 31272388) to JXY and National Natural Science Foundation of China for Talents Training in Basic Science (J1103507). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction in rotifer aging. However, there are few reports of changes during aging in the expression of genes encoding antioxidant enzymes. Reactive oxygen species (ROS), such as hydrogen peroxide 2 The rotifer Brachionus calyciflorus has a short lifespan (5–7 days) and (H2O2) and superoxide anion (O2 ), are produced mainly as by- it is easy to detect changes in gene expression during the aging products of aerobic respiration and can damage many cell process in this species. Therefore, we analyzed changes in the macromolecules, including lipids, proteins and nucleic acids [1]. mRNA expression level of genes encoding the antioxidants This damage has been hypothesized to be the major contributor to manganese SOD (MnSOD) [14], copper and zinc SOD the aging process [1–4]. However, organisms have well-developed (CuZnSOD) and CAT (GenBank accession no. JX000005, defense systems to eliminate ROS [5]. For example, antioxidant JX515577 and JX515578, respectively) in B. calyciflorus individuals enzymes, such as superoxide dismutase (SOD) and catalase (CAT), of different ages to explore the role of such enzymes in the aging catalyze the decomposition of ROS [6,7], thereby moderating process. oxidative stresses and resulting in longer lifespan [8]. Previous Dietary restriction (DR) is usually defined as a moderate studies have shown that SOD expression enhancement can extend (normally 20–40%) reduction of available nutrients without the lifespan of the nematode Caenorhabditis elegans [9] and the fruit causing malnutrition. It was first noted during the 1930s that fly Drosophila melanogaster [10]. Rotifers are important members of DR significantly extended the lifespan of rodents [15]. In the freshwater zooplankton communities and, because of their laboratory, DR is one of the most commonly used life-history ecological significance, are increasingly being used in ecotoxico- alterations among evolutionarily distinct eukaryotes, from single- logical studies [11,12]. In addition, they are often used in aging cell to multicellular organisms [16–19]. Previous studies have research, because of their small body size (0.1–1 mm in length) suggested that DR can extend the lifespan of rotifers and enhance and short lifespan (6–14 days). Studies have also shown that their resistance to oxidative stress [20–24]. Interestingly, DR- antioxidants can extend the rotifer lifespan [13], which suggests induced longevity and other beneficial effects of DR can transmit that genes encoding antioxidant enzymes have an important role

PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e57186 Antioxidant Enzyme Contribute to Delaying Aging to the next generation in rotifers [25]. Resveratrol (3,5,40- Dietary restriction. Rotifers hatched from resting eggs were trihydroxy-trans-stilbene) is a polyphenolic phytoallexin found in cultured for 24 h in normal media. In total, 100 rotifers were then a variety of plant products [26], and has been reported to have selected at random and transferred into 50 mL of DR medium (i.e. beneficial effects on lifespan in many organisms, including yeast no food) and starved for 12, 24 and 48 h. As above, each Saccharomyces cerevisiae [26], D. melanogaster, C. elegans [27,28] and the treatment had three replicates. Rotifers were transferred into fish Nothobranchius furzeri [29]. It also has been reported that newly prepared culture media every 24 h and checked every 12 h resveratrol can protect against ROS-induced cell death by to remove neonates. Rotifers were collected at 12, 24 and 48 h and inhibiting ROS formation [30], preventing mitochondrial dys- the total RNA was then extracted. function [31] and restoring SOD activity [32]. Oxidative stress resistance in rotifers pretreated with DR The objective of present study was to investigate the effects of or resveratrol. Rotifers hatched from resting eggs were DR and resveratrol on the expression levels of the genes encoding pretreated by either DR (i.e. provided with food once every MnSOD, CuZnSOD and CAT. Lifespan extension is always 12 h) or resveratrol (5 mM), and were then transferred into accompanied by an increase in viability in wide variety of species. paraquat (15.25 mg/L) media. Ten rotifers were added to six-well Therefore we exposed the rotifers pretreated with DR and culture plates that contained 5 mL of the test solution. Each resveratrol to oxidative stress, and analyzed their survival. treatment had three replicates. The number of rotifers surviving after 12 h and 24 h was recorded. Resveratrol was dissolved in Materials and Methods acetone and stored at 220uC. Paraquat (DMSO stock) was obtained from the Jiangsu Academy of Agricultural Sciences Rotifers (Nanjing, China). The used during the study, Brachionus calyciflorus (Pallas), Age-related changes in swimming speed. To measure the were neonate females (0–2-h old) hatched from resting eggs in rotifer swimming speed, we modified the method of Snell et al. artificial freshwater medium (EPA medium, pH ,7.8) that (2011) as follows [13]. Experiments were carried out in a 48-well comprised 96 mg NaHCO3, 60 mg CaSO4?H2O, 123 mg plate (10.20 mm in diameter) that contained 100 mL EPA. Video MgSO4, and 4 mg KCl in 1 L deionized water at 25uC [33]. footage of rotifers swimming at 64 magnification was captured Members of this species were originally collected in Gainesville, with a camera attached to a LEICA EZ4D stereomicroscope. Florida in 1983 [34] and, since then, have been cultured AutoCAD 2011 software was used to estimate the swimming speed continuously in the laboratory with periodic collection and storage (mm/s) of individual rotifers. From each age groups (0, 1, 2, 3, 4 of resting eggs. The females were fed by suspending the green alga and 5 days), eight to ten randomly selected rotifers were each Chlorella pyrenoidosa (Institute of Hydrobiology, Chinese Academy of videoed swimming for at least 1 min to measure the swimming 6 Sciences, Wuhan, China) in the culture media at ,3610 cells per speed. mL. The feeding alga was cultured in BBM (Bolds Basal Medium) Quantitative analysis of gene transcription. Total RNAs medium at 25uC in 3 L flasks under constant fluorescent were extracted using the procedures described above. Total RNA illumination at 2000 lux. was reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit (TaKaRa, Japan). Real-time PCR was performed on Experimental design a Mastercycler Ep Realplex (Eppendorf, Germany) to study the Age-related changes in antioxidant enzyme gene gene expression. The PCR reaction was performed in a 25 mL expression. In total, 600 neonates (0–2-h old) were collected volume with a SYBR Premix Ex TaqTMKit (TaKaRa, Japan), and divided into six groups of 100 individuals, which were then 2 mM of each specific primer, and 1 mL of cDNA using the cultured in 50 mL of prepared media maintained at 25uC for 0.5, following procedure: initial denaturation at 95uC for 2 min 1, 2, 3, 4 and 5 days. Each treatment had three replicates. Rotifers followed by 40 cycles of amplification (95uC for 10 s and 55uC were transferred into new medium every 24 h, and checked every for 30 s). Table 1 shows the primers used in the quantitative real- 12 h to record the number of neonates, and the neonates were time PCR. The gene encoding b actin (GenBank accession no. removed each day. Any surviving rotifers were then collected after JX441322) was used as the internal control. The relative 0.5, 1, 2, 3, 4 and 5 days. The total RNA was then extracted and expression levels of the different genes were calculated using the reverse-transcribed into cDNA. Real-time PCR was carried out to 22DDCT method [36]. measure the expression levels of MnSOD, CuZnSOD and CAT at different points of time above. The real-time PCR method was as described below. Culture in 5 mM resveratrol. In total, 500 rotifers (0–2 h Table 1. Quantitative real-time PCR primers used in the old) were collected and divided into five groups of 100 individuals, experiment. which then cultured in 50 mL of resveratrol (5 mM) media. As above, each treatment had three replicates. Culture plates were Primers Sequence (5’–3’) maintained at 25uC in darkness. The same number of rotifers was cultured in normal media as a control. As above, rotifers were SOD-RTF TTGCTTGTTCAAAACATACTC transferred into new resveratrol medium every 24 h, checked SOD-RTR TAATTGCTTCAGCTAAGTCTCC every 12 h to record the number of neonates, and the neonates CuZn SOD -RTF GCCTTGACTCTCCTGTAC were removed each day. Rotifers were collected at 12, 24, 48, 72 and 96 h and the total RNA was then extracted. Resveratrol was CuZn SOD -RTR GCTCGTCCTATTATAGAATGTG obtained from Sigma Chemical Company (St. Louis, MO, USA), CAT-RTF ACAAGGCAATCCAGTCTATT and was dissolved in acetone at a concentration of 50 mM and CAT-RTR AATCGTCCAACAGGTATCAA stored at 220uC. Final concentrations of resveratrol in the test b Actin-RTF GAAATTGTGCGCGACATCAAGGA medium were 5 mM and the concentration of acetone was 0.25%. b Actin-RTF GCAATGCCCGGGTACATGGTGGT This concentration of acetone had no detectable physiological effect on the rotifers [35]. doi:10.1371/journal.pone.0057186.t001

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Statistical analysis lengthened in some individuals. Swimming speed also changed Data were collected that represented the mean 6 standard error significantly with the aging process (Figure 3). Newly hatched (S.E.). All the statistical analyses were carried out with the SPSS rotifers swam at 4.3-body lengths/s (,0.78 mm/s). On the second 16.0 analytic package. Statistical significance was determined by day, the swimming speed peaked (1.89 mm/s, ,6.3-body lengths/ one-way analysis of variance (ANOVA) and post-hoc Duncan s) and then began to slowly decline. multiple range tests. Significance was set at P,0.05. All the figures were produced using Sigma Plot version 11.0. Age-related changes in antioxidant enzyme gene mRNA expression Results The changes in antioxidant enzyme gene expression are illustrated in Figure 4. The mRNA expression level of MnSOD Aging-related changes in rotifer phenotype decreased with aging (P,0.05), whereas that of CuZnSOD An example of a B. calyciflorus survivorship curve and fecundity increased significantly (P,0.05). The expression of CAT did not schedule under control conditions is shown in Figure 1. Under change significantly (P.0.05). these experimental conditions (25uC, ad libitum food), animals began reproducing after a short juvenile period of 18–20 h. Each Egg-related changes in antioxidant enzyme gene mRNA rotifer was able to produce at least one generation and to carry no more than two unfertilized eggs 24 h post-hatching. Reproduction expression peaked at the age of 2 days, and was recorded as being In rotifers carrying at least three eggs, MnSOD and CAT approximately three offspring per female per day. Each female mRNA expression levels were significantly higher (P,0.05); by produced an average of 15.5 offspring in her lifetime. Under these contrast, the mRNA expression of CuZnSOD decreased (P,0.05; conditions, rotifer reproduction was exclusively parthenogenetic. Figure 5). The first death occurred at 72 h, and numbers gradually increased with increasing age of the remaining rotifers. Under normal The effects of resveratrol and DR on antioxidant enzyme experimental conditions, the mean rotifer lifespan was 102 h. gene mRNA expression Figure 2 illustrates the aging phenotypes over the B. calyciflorus Quantitative real-time PCR showed that resveratrol significant- lifespan. Newly hatched rotifers were ,180 mm long and grew to ly increased (P,0.05) the mRNA expression of the gene encoding ,300 mm in length after ,12 h, when they matured into adults. MnSOD (Figure 6a), whereas the expression levels of the genes This increase in size is known to be the result of water uptake and encoding CuZnSOD (Figure 6b) and CAT (Figure 6c) were cytoplasm synthesis without cell division, given that rotifers are inhibited. The mRNA expression of MnSOD was enhanced after eutelic [13]. The study rotifers usually carried 1 egg at 1-day-old, 24-h DR, but was significantly lower (P,0.05) after 48-h DR and three or more (4–6) eggs at 2–3-days old. After the fourth day, (Figure 7). Starvation also increased the mRNA expression of the their reproduction began to decrease. Egg production is a genes encoding CuZnSOD and CAT, which peaked after 12-h prominent feature of females because eggs are carried at the DR (Figure 7). posterior of the females until they hatch [13]. On the fifth day, the rotifers generally did not reproduce, and their morphology changed significantly; for example, the spines on the posterior

Figure 1. Typical survival and fecundity curves for B. calyciflorus under control conditions. doi:10.1371/journal.pone.0057186.g001

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Figure 2. Photomicrographs of female rotifers of various ages. doi:10.1371/journal.pone.0057186.g002

Effect of DR and resveratrol on rotifer swimming speed at Effect of DR and resveratrol pretreatment on oxidative 4-days-old stress resistance Resveratrol treatment did not increase rotifer swimming speed Figure 9 illustrates the effects of DR and resveratrol pretreat- of 4-days-old rotifers, whereas moderate DR (12 h) increased the ments on the survival rate of rotifers under oxidative stress swimming speed (P,0.05). In the groups starved for 24 h and conditions. After being exposed to oxidative stress for 12 h, the 48 h, there was no significant change in swimming speed (P.0.05; survival rate of the DR pretreated group was 0.7860.02, Figure 8). significantly higher (P,0.05) than of the control group

Figure 3. B. calyciflorus swimming speed (mm/s). doi:10.1371/journal.pone.0057186.g003

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Figure 4. Relative mRNA expression levels of MnSOD, CuZnSOD and CAT from the B. calyciflorus at various ages. Each group contained 100 individuals. Bars represent standard errors. The expression levels were statistically analyzed by one-way ANOVA with the Tukey test. (*P,0.05). doi:10.1371/journal.pone.0057186.g004

(0.6060.25). A similarly significant result was recorded for the Discussion group exposed for 24 h: the survival rates of the DR pretreated and control groups were 0.5560.02 and 0.3860.02, respectively The accumulation of ROS, which are the main by-products of (P,0.05). This result indicates that DR pretreatment can increase aerobic respiration, impairs the function of cellular macromole- the rotifer survival rate under paraquat-induced oxidative stress. cules, including lipids, proteins and DNA, and is considered to be However, resveratrol pretreatment did not significantly improve one of the main causes of aging [1]. Animals have evolved defenses survival under oxidative stress. against ROS damage, and the balance between oxidative stress and concentration of available antioxidants has a major influence on lifespan [13]. Previous studies have demonstrated that high expression of SODs could extend the lifespan of several organisms

Figure 5. Relative mRNA expression levels of MnSOD, CuZnSOD and CAT from female B. calyciflorus carrying different numbers of eggs. Bars represent standard errors. Egg 0, Egg 1, Egg 2, Egg 3 and Egg .3 represent rotifers carry 0, 1, 2, 3 and 4 or more eggs, respectively. The expression levels were statistically analyzed by one-way ANOVA with the Tukey test. (*P,0.05). doi:10.1371/journal.pone.0057186.g005

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[10,37]. Although the mechanism of antioxidant effects on aging remains controversial [38], the ability of antioxidants to extend rotifer lifespan has been confirmed [13]. In this study, we investigated how antioxidant enzyme gene mRNA expression changes with age, and our results are likely to be useful for further studies on the role of antioxidant genes in aging. The mRNA expression levels of MnSOD decreased with aging, whereas those of CuZnSOD increased. However, the mRNA expression level of CAT did not change significantly. MnSOD (SOD, E.C. 1.15.1.1) 2 is a mitochondrial protein that catalyzes the conversion of O2 to the less reactive H2O2 [6]. MnSOD is considered to be the most important isoform for the regulation of lifespan, because mitochondria are the main source of ROS [24]. Therefore, decreases in MnSOD expression are not conducive to the elimination of ROS. CAT (E.C. 1.11.1.6), another antioxidant enzyme containing heme, catalyzes the degradation of H2O2 to water (H2O) and molecular oxygen (O2) [24,39]. CAT is also an enzyme with one of the highest turnovers and is important in reproductive reactions. CuZnSOD, which is localized in cyto- plasm, contains copper and zinc at the catalytic centers and also 2 catalyzes O2 to H2O2 [6]. Given that ROS are mainly produced inside organelles, such as mitochondria [40,41], such cellular bodies are among the first to be damaged by ROS. In addition, accumulating oxidative damage can affect the efficiency of mitochondria and further increase the rate of ROS production [42]. Thus, increased expression of MnSOD and CAT retards the aging process and extends lifespan, as shown by several overexpression studies in D. melanogaster [43,44] and mouse Mus musculus [45]. The reduction of MnSOD expression level with age will aggravate ROS-induced mitochondrial damage. Although CuZnSOD expression increased with age, it can only have an effect in the cytoplasm. Snell and co-workers measured the concentration of ROS in rotifers of different ages and found no increase in ROS load with aging [13]. Therefore, ROS damage accumulation might be a major contributor to the aging process [1–4,46]. It is a widespread phenomenon that many organisms, including C. elegans [47], D. melanogaster [48] and rodents [49,50] would increase their lifespan and/or reduce their reproductive rate when food is less available [51]. Therefore, there is likely to be a close relationship between aging and reproductive effort. Lifespan elongation and fecundity reduction was also observed when rotifers were denied food [22,51,52], which suggests that suspension or slowdown of reproduction can retard the rate of aging process and prolong lifespan. The present study shows that in rotifers carrying three or more eggs, MnSOD and CAT expression levels were significantly higher (Figure 5). This result suggests that reproduction increases oxidative stress and influences the expression of antioxidant enzyme genes. This would be a reason that accelerates the aging process of rotifers. However, the relation between reproduction and aging is intricate and requires further study as to whether aging is mainly or directly caused by reproduction in rotifers. DR is a well-known method of increasing both lifespan and resistance to exogenous oxidative stress provoked by ROS generators [53]. In rotifers, DR influences the expression of many genes, including those encoding SOD [8,25], CAT [25], heat Figure 6. Relative mRNA expression levels of genes encoding shock protein (HSP)-70 and Glucose-regulated protein (GRP)-94 the antioxidant enzymes. (a) manganese superoxide dismutase [54], and extends lifespan [20–22,55]. In our study, we found that (MnSOD), (b) copper and zinc SOD (CuZnSOD) and (c) catalase (CAT) moderate DR (12 and 24 h) increased the expression of the genes from B. calyciflorus rotifers treated with resveratrol. The concentration of encoding MnSOD, CuZnSOD and CAT (Figure 7). This resveratrol was 5 mM/L. Rotifers cultured in normal medium were used as the control. Each group contained 100 individuals. Bars represent antioxidant gene expression enhancement could, to some extent, standard errors. The expression levels were statistically analyzed by one- explain why DR extends lifespan. However, aging is likely to be way ANOVA with the Tukey test. (*P,0.05). regulated by multiple, low-affinity, low-specificity interaction

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Figure 7. Relative mRNA expression levels of MnSOD, CuZnSOD and CAT from B. calyciflorus under various DR conditions. AL, DR12, DR24 and DR48 represent rotifer groups starved for 0, 12, 24 and 48 h, respectively. Bars represent standard errors. The expression levels were statistically analyzed by one-way ANOVA with the Tukey test. (*P,0.05). doi:10.1371/journal.pone.0057186.g007 processes, mediated through a complex tangle of overlapping scavenger and regulates SOD expression in nematodes [60] and pathways [13]. Antioxidant genes might be just one part of the rats [32]. In our study, resveratrol did not extend the lifespan of whole system. the rotifers (data not shown), although it did induce MnSOD Many of the polyphenols such as resveratrol and quercetin, are expression, the high levels of which might contribute to reducing synthesized by plants under different stresses (e.g. infection, the damage caused by ROS in mitochondria [61]. However, starvation or dehydration) [53,56]. Resveratrol has beneficial resveratrol might inhibit CuZnSOD and CAT expression, thus effects on lifespan in a range of organisms, from yeast to mammals resulting in aggravating ROS damage to other organelles. This [26–29,57,58], through a DR-like mechanism. This is dependent might explain why resveratrol cannot extend the lifespan of on an NAD+-dependent histone deacetylase, Sir2 in Drosophila and rotifers. Moreover, resveratrol did not improve the swimming SIR-2.1 in C. elegans [59]. Resveratrol acts as a strong radical speed of rotifers (at 4-days-old), which further confirms the fact

Figure 8. The swimming speed of 4-days-old B. calyciflorus. AL, DR12, DR24 and DR48 represent the rotifer groups starved for 0 h, 12 h, 24 h and 48 h, respectively. Bars represent standard errors. The asterisk indicates significant difference from control. (*P,0.05). doi:10.1371/journal.pone.0057186.g008

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To some extent, high levels of antioxidant enzyme gene expression might convey greater resistance to oxidative stress. To assess the resistance to oxidative stress in rotifers, we used paraquat, a widely used compound that generates ROS inside the cell [25]. Under this oxidative stress, DR pretreatment increased the rotifer survival rate. However, resveratrol pretreatment had no effect on rotifer survival rate under oxidative stress. DR treatment increased the expression of genes encoding all three antioxidant enzymes (MnSOD, CuZnSOD and CAT), whereas resveratrol treatment only increased expression of the gene encoding MnSOD. We believe that inducing the expression of genes encoding antioxidant enzymes would contribute to improving the ability of an organism to resist oxidative stress. In this study, we examined the effects of aging on the expression of genes encoding antioxidant enzymes in rotifers. Our results suggest that the decrease in antioxidant gene expression is one of the causes of aging. Lifespan extension by DR might also be associated with an increase in oxidation resistance. Resveratrol, as an exogenous antioxidant, can facilitate the expression of MnSOD only, although its inhibition of CuZnSOD and CAT might be one of the reasons why lifespan did not extend in those rotifers treated with this compound.

Figure 9. The survival rate of B. calyciflorus under oxidative Acknowledgments stress conditions (paraquat). The survival rate was statistically We would like to thank Yawen Mu of China Southeast University for analyzed by one-way ANOVA and post-hoc Duncan multiple range test. improving the manuscript. The different letters indicate a significant difference for each time point with control (P,0.05). doi:10.1371/journal.pone.0057186.g009 Author Contributions Revised the paper: YWM SMD. Conceived and designed the experiments: that resveratrol has no effect on the rotifer aging process (Figure 8). JXY. Performed the experiments: JHY. Analyzed the data: JHY QCJ. Snell and co-workers found that dietary exposure to only certain Contributed reagents/materials/analysis tools: SMD WTH TJK. Wrote antioxidants extended rotifer lifespan [13]. This phenomenon the paper: JHY. suggests that the mechanism of antioxidant action is still unclear.

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PLOS ONE | www.plosone.org 9 February 2013 | Volume 8 | Issue 2 | e57186